JP3115289B2 - Plant pathogen control agent using chitinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an agent for controlling phytopathogenic bacteria using chitinase, particularly yam-derived chitinase.

[0002]

2. Description of the Related Art Conventionally, as a plant pathogen-controlling agent, an organic synthetic plant pathogen-controlling agent such as a heterocyclic aromatic compound or an organic phosphate ester type has been mainly used. These drugs not only exert their effects on pathogenic bacteria, but also have an adverse effect on the human body and cause problems such as residual pesticides.

[0003] Chitinase is an enzyme that hydrolyzes chitin, and is used for molting of arthropods and crustaceans, defense of plants, growth of microorganisms, and the like. However, the antibacterial properties of chitinase are not known, and chitinase has not been used as a plant pathogen control agent. On the other hand, chitinase is considered to be safe for the human body and the environment because it is of biological origin.

[0004]

As described above, various problems have been pointed out with respect to the phytopathogenic fungi using the organic synthetic compound. For this reason, a novel safe plant pathogen control agent has been desired to replace such a type of plant pathogen control agent. An object of the present invention is to provide a novel plant pathogen control agent utilizing a substance derived from an organism in order to meet such a demand.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that chitinase has antibacterial activity and that the antibacterial effect of chitinase is β-1,3-
Found to be synergistically increased by glucanase,
The present invention has been completed. That is, the present invention is an agent for controlling phytopathogenic bacteria, comprising chitinase as an active ingredient. The present invention also relates to chitinase and β
It is a plant pathogen control agent comprising -1,3-glucanase as an active ingredient.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Chitinase used in the present invention is not particularly limited,
Preferred chitinases include yam-derived chitinases, and among them, particularly preferred chitinases include Yam H1 and Yam H2. Yam H1 is a chitinase isolated by the present inventors, whose optimum pH is 4 (for chitin oligosaccharide), 3 and 9 (for glycol chitin), and stable pH range is 3-12. The optimum temperature is 70 ° C, and the stable temperature range is 0 to 80 ° C. Considering that a conventionally known chitinase, for example, a rye-derived chitinase has a stable pH range of 4 to 8 and a stable temperature range of 0 to 50 ° C., Yam H1 is extremely excellent in heat and pH stability. I can say. This chitinase contains at least the amino acid sequence of SEQ ID NO: 1. Ya
mH2 is also a chitinase isolated by the present inventors, and has at least the amino acid sequence of SEQ ID NO: 2.

The plant pathogen controlling agent of the present invention may contain chitinase alone as an active ingredient, but more preferably chitinase and β-1,3-glucanase. The β-1,3-glucanase used in the present invention may be derived from a plant or a microorganism, and is not limited to a specific organism.

As the phytopathogenic fungicide of the present invention, chitinase and β-1,3-glucanase may be used as they are, but they are generally dissolved or dispersed in a suitable liquid carrier, or mixed with a suitable powder carrier. Emulsifiers, oils, wettable powders, etc., if necessary, by mixing or adsorbing them, and adding emulsifiers, dispersants, suspending agents, spreading agents, penetrants, wetting agents, stabilizers, etc. , Powders and the like.

[0008] The liquid carrier used in the preparation includes water, phosphate buffer, aqueous solution of organic acids, liquid fertilizer, seaweed / plant / wood extract, aqueous solution of minerals, aqueous solution of amino acids,
An aqueous solution of Chinese herbs is suitable, and one or more of these can be used in combination.

[0009] Further, as a powder carrier used in the preparation,
Clay minerals such as talc, clay and pyrophyllite, activated carbon, zeolite, silica, cellulose, calcium, chitosan,
Starch and the like are suitable, and one or more of these can be used as a mixture.

[0010] In the case of a plant pathogen control agent characterized by containing chitinase as an active ingredient, the concentration of chitinase as an active ingredient is generally 0.01 to 0.05 in the case of an emulsion.
%, 0.005 to 0.025% for fats and oils, 0.01 to 0.05% for wettable powders, and 1.0 to 5.0% for powders, and these concentrations may be changed as appropriate.

In the case of a phytopathogenic fungicide comprising chitinase and β-1,3-glucanase as active ingredients, the active ingredients chitinase and β-1,3
-The concentration of glucanase is in each case from 0.01 to
0.05%, 0.01 to 0.05%, 0.005 to 0.02 for fats and oils
5%, 0.005-0.025%, 0.01-0.05 for wettable powder
%, 0.01-0.05%, in the case of dust, 0.01-0.05%, 0.01
-0.05% is appropriate, and these concentrations may be appropriately changed.

[0012] The amount of the phytopathogenic fungicide characterized by containing chitinase as an active ingredient is per 10a
0.1 ~ 1.0 kg, but 0.1 ~ per 10a
It is preferably 0.5 kg. Also, chitinase and β-1,3
-The amount of the phytopathogenic fungicide characterized by containing glucanase as an active ingredient is 0.1 to 10 per 10a.
1.0kg, but 0.1 ~ 0.5kg per 10a
It is preferred that

The phytopathogenic fungi of the present invention can control phytopathogenic fungi. For example, the phytopathogenic agent of the present invention is Phoma wasabiae.
e), Fusarium ox.sp.raph
ani), Rozelinia necatrix
It exerts an excellent control effect on plant pathogens such as. In particular, in the case of a phytopathogenic fungicide characterized by containing chitinase and β-1,3-glucanase as active ingredients, the plant enhanced by the synergistic effect of chitinase and β-1,3-glucanase A pathogen control effect is exhibited.

[0014]

[Example 1] Induction of chitinase by chitosan Chitosan was added to rice free cells that had been liquid-cultured in advance to a final concentration of 100 µg / ml, and cultured with shaking for 3 days. Thereafter, the cell culture solution was subjected to suction filtration to remove the cells, and a crude enzyme solution was extracted. After adding N-acetylchitosan as a substrate to the crude enzyme solution, the mixture was centrifuged, and the reducing sugar released in the supernatant was quantified by the modified Charles method to measure the chitinase activity. Controls were those cultured without addition and those cultured with addition of only lactic acid used as a solvent for chitosan. As a result, the addition of chitosan to the rice free cell culture increased rice chitinase activity (Table 1). This indicated that chitosan induced chitinase in rice free cells.

[0015]

[Table 1]

Example 2 Determination of the concentration of chitosan that promotes chitinase induction Chitosan having a degree of deacetylation of 80% was added to rice free cells that had been liquid-cultured in advance so that the final concentration was 50, 100, or 1000 μg / ml. And shake-cultured for 3 days. Thereafter, the cell culture solution was subjected to suction filtration to remove the cells, and a crude enzyme solution was extracted. After adding N-acetylchitosan as a substrate to the crude enzyme solution, the mixture was centrifuged, and the reducing sugar released in the supernatant was quantified by the modified Charles method to measure the chitinase activity. As a result, the concentration of 100 μg / ml showed the highest chitinase activity (FIG. 1).

Example 3 Determination of Optimum Temperature and Optimum pH of Rice Chitinase Activity N-acetylchitosan was added to the filtrate containing rice chitinase extracted in the same manner as in Example 1, and the temperature was adjusted to 20, 30, 4
The mixture was allowed to stand at 0, 50, 60, 70, 80, and 90 ° C for 5 minutes. afterwards,
Each filtrate was centrifuged, and the chitinase activity was measured by quantifying the reducing sugar released in the supernatant by the modified Charles method. Also, regarding the pH, the filtrate was adjusted to pH 2, 4
, 6, 8, 10, and 12 were adjusted with 1N HCl and NaOH, and chitinase activity was measured by the modified Charles method in the same manner as described above. As a result, the optimal temperature is 50-60 ° C,
The activity was stable even at relatively high temperatures (FIG. 2). Also,
The optimum pH was 6-7 (FIG. 3).

[Example 4] Antibacterial effect of rice chitinase A chitosan solution having a degree of deacetylation of 80% was added to rice cells previously liquid-cultured to a final concentration of 100 µg / ml, and cultured with shaking for 3 days. did. Thereafter, the cell culture solution was subjected to suction filtration to remove cells, and the filtrate was dialyzed to obtain a crude enzyme solution. A YG agar medium (Yeast. Extract 0.1%, Glucose 0.1%, KH ₂ PO ₄ 0.02%,
K ₂ HPO ₄ 0.03%, MgSO ₄・ 7H ₂ O 0.02%, agar 1.5%) was prepared and pre-cultured pathogenic bacteria (Pyricularia oryzae)
And Phoma wasabiae) were extracted and inoculated with a cork borer having a diameter of 5 mm and cultured. Using the YG medium containing no crude enzyme solution as a control, the area ratio of the pathogenic bacteria grown on the control YG agar medium and the YG agar medium containing the crude enzyme solution was calculated.

As a result, in the case of Pyricularia oryzae, the area ratio between the control YG agar medium and the YG medium containing 10% of the crude enzyme solution was 100: 87.
The area ratio with the YG medium containing 20% was 100: 58, and the control Y
Area ratio between G agar medium and YG medium containing 30% of crude enzyme solution
The ratio was 100: 41 (FIG. 4). In the case of Phoma wasabiae, the area ratio between the control YG agar medium and the YG medium containing 10% crude enzyme solution was 100: 38, and the area ratio between the control YG agar medium and the YG medium containing 20% crude enzyme solution was used. The area ratio was 100: 22, and the area ratio between the control YG agar medium and the YG medium containing 30% of the crude enzyme solution was 100: 12 (FIG. 5).

As described above, the growth of Pyricularia oryzae and Phoma wasabiae was inhibited on the YG agar medium containing the crude enzyme solution, whereby rice chitinase was converted to Pyr
It has been shown to have antibacterial effects against icularia oryzae and Phoma wasabiae.

Example 5 Lysis Activity by Chitinase and β-1,3-Glucanase The mycelium of Fusarium oxysporum grown on a 3.9% potato dextrose agar medium was replaced with 0.4 M MgSO ₄
Plant chitinase (Yamamo chitinase H) and 3% β-protein in MacIlvaine buffer (pH 4.0) containing 5 mM sodium phosphate buffer (pH 8.0) containing 0.4 M mannitol.
1,3-glucanase (Zymolyase®) and 27
When reacted at ℃, protoplasts were released from the mycelium of the pathogenic bacterium (FIG. 6). From this, it was found that the combination of the plant chitinase and β-1,3-glucanase dissolved the cell wall of the pathogenic bacterium and exhibited an antibacterial effect.

Example 6 pH Stability of Yam H1 After incubating Yam H1 in buffers of different pH,
The enzyme activity was measured and the pH stability of Yam H1 was determined. Incubation was performed at 4 ° C. for 15 hours. The enzyme activity was measured at pH 4.0 using GlcNAc ₅ as a substrate. The result is shown in FIG. As shown in FIG. 7, the stable pH range of Yam H1 is considered to be 3-12.

Example 7 Temperature Stability of Yam H1 After Yam H1 was incubated in buffers at different temperatures, the enzyme activity was measured and the temperature stability of Yam H1 was examined.
Incubation was performed at pH 7.0 for 15 minutes. The enzyme activity was measured at pH 4.0 using glycol chitin as a substrate. The result is shown in FIG. As shown in FIG.
It is considered that the stable temperature range of H1 is 0 to 80 ° C.

[Example 8] Lysis activity of yam chitinase H Plant pathogenic fungi, Fusarium oxysporum, Fuzarium roseum,
To 0.25 mg of three types of mycelia of Pyrenophora graminea, 17 μM final concentration of yam chitinase and 3% final concentration of Zymolyase (registered trademark) were added, and pH 8.0, 27
The reaction was performed in a dark place at 14 ° C., and the reaction solution was observed with a microscope after 14 hours. FIG. 9 shows this state. All three types of plant pathogens released protoplasts of about 5 microns, and lytic activity was observed.

Example 9 Purification of Yam Chitinase H Example 9 Isolation of Yam H1 and Yam H2 Yam tuber bark (about 200 g) was homogenized together with three times its volume of 0.1 M phosphate buffer (pH 7.0). . It was centrifuged at 9000g for 20 minutes and the supernatant was collected. In the supernatant, 1/20 volume of 10% CTAB (hexadecyl trimethyl amm
onium bromide) was added dropwise with stirring. The mixed acidic polysaccharide was removed as a precipitate. After standing overnight, the solution was centrifuged at 9000 g for 20 minutes to collect the supernatant, and ammonium sulfate was added thereto to obtain a 60% saturated ammonium sulfate solution, followed by thorough stirring. After one night, this was centrifuged at 9000 g for 20 minutes to collect the precipitate, which was dialyzed against 10 mM phosphate buffer (pH 8.0). This dialysate was equilibrated with the same buffer as the dialysate
It was applied to a DEAE-Cellulofine A-500 column (2.2 × 35 cm).
After elution with the same buffer until no protein was present, the chitinase was eluted with a 0 M to 0.5 M sodium chloride gradient in the same buffer. Chitinase H eluted in the fraction with a conductivity of 5-10 mmho. This was further combined with the same DEAE-CellulofineA-500 column (2.2x35 c
m), two kinds of chitinases were separated.
Named H1 and Yam H2.

Yam H1 was dialyzed against 25 mM histidine-hydrochloric acid buffer (pH 5.5), and a column for chromatofocusing, polybuffer-exchange column (1 × 20 c)
m) Called. Elution was performed using a solution prepared by diluting polybuffer-74 eight-fold and adjusting the pH to 3.0. Yam H1 eluted at pH 3.6 (isoelectric point 3.6). This Yam H1 fraction was dialyzed against 50 mM phosphate buffer containing 0.1 M sodium chloride, and then equilibrated with the same buffer in a Cellulofine GC-200 column (2 × 120 c).
Gel filtration was performed in m). It eluted at a molecular weight of about 50,000. The molecular weight was confirmed by SDS-PAGE.
It was found that 00 proteins formed a dimer.
The completion of the purification was confirmed by a single band on native PAGE.

Example 10 Determination of Partial Amino Acid Sequences of Yam H1 and Yam H2 0.3 g of yam leaves were frozen at -80 ° C. and ground with dry ice. To dissolve cells and cell contents, add 1.7 ml of Regent B, mix well, and add RNase to 40 ml.
0 ng / ml was added and incubated at 37 ° C. for 30 minutes. Thereafter, in order to remove unnecessary proteins, 500 μl of 5M sodium perchlorate was added, and the mixture was stirred well at room temperature for 20 minutes. The mixture was incubated for 20 minutes while inverting and mixing several times in a water bath at 65 ° C. After incubation, 2.
9 ml of chloroform was added, the mixture was stirred at room temperature for 20 minutes, and then centrifuged (room temperature, 4000 rpm, 1 minute). After adding 225 μl of Nucleon Silica Suspension to the obtained supernatant, the mixture was centrifuged again (room temperature, 4000 rpm, 1 minute). Only the aqueous layer containing DNA was taken out of the supernatant, and 4.4 ml of cold 100% ethanol was added thereto, followed by gentle stirring. Centrifugation (4
Temperature, 7000 rpm, 5 minutes) to collect the precipitate,
5.0 ml of ethanol was added, followed by gentle stirring. The precipitate was collected and dissolved in 75 μl of TE to obtain yam-derived genomic DNA.

The genomic DNA derived from yam extracted above
Was used as a template to perform PCR. As a primer, Yam
N-terminal side (WGQNGFE) and C-terminal side (NNPPC
An oligonucleotide encoding the amino acid sequence of H) was used. The amplification conditions were 94 ° C for 1 minute, (94 ° C for 30 seconds, 60 ° C for 1 minute).
Min, 72 ° C for 2.5 minutes) 25 cycles, 72 ° C for 7 minutes. As a result of the reaction, a fragment of about 1100 bp was amplified. A part of the base sequence of the fragment was determined, and the amino acid sequence was deduced from the sequence (SEQ ID NO: 1 and SEQ ID NO: 2).

[0029]

According to the present invention, phytopathogenic bacteria can be controlled without adversely affecting the human body and the environment. In particular, in the case of a phytopathogenic fungicide characterized by containing chitinase and β-1,3-glucanase as active ingredients, the plant enhanced by the synergistic effect of chitinase and β-1,3-glucanase A pathogen control effect is exhibited.

[0030]

[Sequence List] SEQUENCE LISTING <110> SANIN KENSETSU KOUGYOU KABUSHIKI GAISYA <120> KICHINAHZE WO MOCHIITA SYOKUBUTSU BYOUGENKIN BOUJYOZAI <130> P99-0421 <160> 2 <170> PatentIn version 2.0 <210> 1 <211> 183 <212> PRT <213> Dioscorea opposita Thunb <400> 1 Trp Gly Gln Asn Gly Phe Glu Gly Ser Leu Ala Glu Ala Cys Ser Thr 1 5 10 15 Arg Asn Tyr Asp Ile Val Val Ile Ala Phe Leu Tyr Gln Phe Gly Asn 20 25 30 Phe Gln Thr Pro Gly Leu Asn Leu Ala Gly His Cys Asn Pro Ala Phe 35 40 45 Gly Gly Cys Val Ser Ile Gly Asn Asp Ile Lys Ala Cys Gln Ser Gln 50 55 60 Gly Ile Lys Val Phe Leu Ser Leu Gly Gly Ala Tyr Gly Ser Tyr Thr 65 70 75 80 Leu Val Ser Thr Gln Asp Ala Gln Gln Val Ala Asp Tyr Leu Trp Asn 85 90 95 Asn Phe Leu Gly Arg Ser Ser Ser Ser Arg Pro Leu Gly Asp Ala Val 100 105 110 Leu Asp Gly Ile Asp Phe Asp Ile Glu Gly Gly Thr Thr Gln Tyr Trp 115 120 125 Asp Glu Leu Ala Gln Met Leu Phe Asp Tyr Ser Gln Gln Gly Gln Lys 130 135 140 Val Tyr Leu Ser Ala Ala Pro Gln Cys Pro Tyr Pro Asp Ala Trp Met 145 150 1 55 160 Gly Lys Ala Leu Ala Thr Gly Leu Phe Asp Tyr Val Trp Val Gln Phe 165 170 175 Tyr Asn Asn Pro Pro Cys His 180 <210> 2 <211> 183 <212> PRT <213> Dioscorea opposita Thunb <400> 2 Trp Gly Gln Asn Gly Phe Glu Gly Ser Leu Ala Glu Ala Cys Ser Thr 1 5 10 15 Arg Asn Tyr Asp Ile Val Val Ile Ala Phe Leu Tyr Gln Phe Gly Asn 20 25 30 Phe Gln Thr Pro Gly Leu Asn Leu Ala Gly His Cys Asn Pro Ala Phe 35 40 45 Gly Gly Cys Val Ser Ile Gly Asn Asp Ile Lys Ala Cys Gln Ser Gln 50 55 60 Gly Ile Lys Val Phe Leu Ser Leu Gly Gly Ala Tyr Gly Ser Tyr Thr 65 70 75 80 Leu Val Ser Thr Gln Asp Ala Gln Gln Val Ala Asp Tyr Leu Trp Asn 85 90 95 Asn Phe Leu Gly Arg Ser Ser Ser Ser Arg Pro Leu Gly Asp Ala Val 100 105 110 Leu Asp Gly Ile Asp Phe Asp Ile Glu Gly Gly Thr Thr Gln His Trp 115 120 125 Asp Glu Leu Ala Gln Met Leu Phe Asp Tyr Ser Gln Gln Gly Gln Lys 130 135 140 Val Tyr Leu Ser Ala Ala Pro Gln Cys Pro Tyr Pro Asp Ala Trp Met 145 150 155 160 Gly Lys Ala Leu Ala Thr Gly Leu Phe Asp Tyr Val Trp Val Gln Phe 165 170 175 Tyr Asn Asn Pro Pro Cys His 180

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the concentration of added chitosan and chitinase activity in rice free cells.

FIG. 2 is a diagram showing the relationship between temperature and chitinase activity.

FIG. 3 is a graph showing the relationship between pH and chitinase activity.

FIG. 4 is a photograph showing the antibacterial effect of chitinase on pathogenic bacteria.

FIG. 5 is a photograph showing the antibacterial effect of chitinase on pathogenic bacteria.

FIG. 6 is a graph showing the lytic activity of chitinase and β-1,3-glucanase.

FIG. 7 is a graph showing the relationship between the pH during incubation and the enzyme activity of Yam H1.

FIG. 8 is a graph showing the relationship between the temperature during incubation and the enzyme activity of Yam H1.

FIG. 9 is a photograph showing the inhibitory effect of Yam H1 on growth of Fusarium bacteria.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-2-502513 (JP, A) JP-A-3-15393 (JP, A) European Patent Application Publication 497865 (EP, A1) Biol. Chem. , Vol. 28, (1992), p. 19944-7 Arch. Biochem. Biops. , Vol. 335, no. 1, (1996), p. 118-122 (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 63/00-63/04 C12N 9/00-9/99 C12N 15/00-15/90 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (G ENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. An agent for controlling phytopathogenic bacteria, comprising as an active ingredient Yam H1 having the following properties. (1) The stable pH range is 3 to 12 (2) The stable temperature range is 0 to 80 ° C. (3) Contains the amino acid sequence of SEQ ID NO: 1 2. Ya comprising the amino acid sequence of SEQ ID NO: 2.
An agent for controlling phytopathogenic bacteria, comprising mH2 as an active ingredient.